An Allosteric Signaling Pathway of Human 3-Phosphoglycerate Kinase from Force Distribution Analysis

Balog E (2014) An Allosteric Signaling Pathway of Human 3-Phosphoglycerate Kinase from Force Distribution Analysis. PLoS Comput Biol 10(1): e1003444. doi:10.1371/journal.pcbi.1003444 An Allosteric Signaling Pathway of Human 3- Phosphoglycerate Kinase from Force Distribution Analysis Zoltan Palmai 0 Christian Seifert 0 Frauke Gra ter 0 Erika Balog 0 Guanghong Wei, Fudan University, China 0 1 Department of Biophysics and Radiation Biology, Semmelweis University , Budapest, Hungary, 2 Molecular Biomechanics , Heidelberger Institut fu r Theoretische Studien gGmbH , Heidelberg, Germany , 3 MPG-CAS Partner Institute and Key Laboratory for Computational Biology , Shanghai , China 3-Phosphogycerate kinase (PGK) is a two domain enzyme, which transfers a phosphate group between its two substrates, 1,3-bisphosphoglycerate bound to the N-domain and ADP bound to the C-domain. Indispensable for the phosphoryl transfer reaction is a large conformational change from an inactive open to an active closed conformation via a hinge motion that should bring substrates into close proximity. The allosteric pathway resulting in the active closed conformation has only been partially uncovered. Using Molecular Dynamics simulations combined with Force Distribution Analysis (FDA), we describe an allosteric pathway, which connects the substrate binding sites to the interdomain hinge region. Glu192 of alpha-helix 7 and Gly394 of loop L14 act as hinge points, at which these two secondary structure elements straighten, thereby moving the substrate-binding domains towards each other. The long-range allosteric pathway regulating hPGK catalytic activity, which is partially validated and can be further tested by mutagenesis, highlights the virtue of monitoring internal forces to reveal signal propagation, even if only minor conformational distortions, such as helix bending, initiate the large functional rearrangement of the macromolecule. - Funding: We thank the Graduate College 850 (University of Heidelberg, Germany; DFG funded), Klaus Tschira Foundation (Heidelberg, Germany) and the European Unions Seventh Framework Programme (FP7/2007-2013) under grant agreement nu HEALTH-F2-2011-278850 (INMiND) for financial support. EB is thankful for the Janos Bolyai Research Scholarship provided by the Hungarian Academy of Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 3-Phosphoglycerate kinase (PGK) is a key enzyme in glycolysis that catalyzes phospho-transfer from 1,3-bisphosphoglycerate (BPG) to ADP producing 3-phosphoglycerate (PG) and ATP [1]. It has been shown that human PGK (hPGK) also phosphorylates L-nucleoside analogues, which are potential antiviral and anticancer drugs [26]. PGK is a monomeric enzyme composed of two domains of approximately equal size with the C-terminus of the protein bending back to the N-terminal domain, constituting an integral part of it. BPG binds to the N-terminal domain while the ADP binding site is located on the C-terminal domain (for the nomenclature of the secondary structure elements see Suppl. Table S1.) Crystal structures of PGK from numerous species have shown the enzyme in two distinct conformations: the open conformation [712], where the substrate binding sites are too far from each other (1215 A) for the phosphoryl transfer reaction to occur (Figure 1), and the closed conformation [1315], where the substrates are proximal enough to allow nucleophilic attack. Thus, these end states are experimental evidence for a hinge bending motion of the enzyme that brings the substrates of this bimolecular reaction together. From combined crystallographic data and small angle X-ray scattering, it has been hypothesized that a spring loaded trap and release mechanism regulates the opening and closing of the domains [12]. By normal mode analysis of the open structure of PGK, Guilbert et al. [16] described three types of interdomain motions: hinge bending, twisting and a shear motion. Our previous Molecular Dynamics (MD) simulations showed that both the apo and the complex enzyme exhibit a small amplitude hinge bending type of motion on nanosecond scale, with the substrate binding changing the character of the motion and restraining the hinge bending [17]. These data put forward the notion of PGK being able to exhibit fast small amplitude hinge bending motions even in the apo state. However, full closure of the enzyme upon substrate binding to adopt the active conformation requires a large and directional hinge bending motion. This raises the question of how the signal of substrate binding penetrates to the interdomain region, where the conformational change happens, leading to closing/opening of the enzyme. Szabo et al. [18] in their combined mutational and kinetic experimental work identified amino acids that play a role in the communication between the substrate binding sites and the hinge region of the enzyme, in particular reported Arg38, Lys219, Asn336 and Glu343 as residues essential in domain closure. A coherent signaling pathway from the binding sites to the hinge region of hPGK has, however, remained elusive. Computational methods that have been developed to describe allosteric communication are mostly based on coordinates, among others by following the correlated motions as observed in Molecular 3-Phosphoglycerate kinase (PGK) is an essential enzyme for living organisms. It catalyzes the phospho-transfer reaction between two catabolites during carbohydrate metabolism. In addition to this physiological role, human PGK has been shown to phosphorylate L-nucleoside analogues, potential drugs against viral infection and cancer. PGK is a two domain enzyme, with the two substrates bound to the two separate domains. In order to perform its function the enzyme has to undergo a large conformational change involving a hinge bending to bring the substrates into close proximity. The allosteric pathway from the open nonreactive state of PGK to the closed reactive state as triggered by substrate binding has only been partially uncovered by experimental studies. Here we describe a complete allosteric pathway, which connects the substrate binding sites to the interdomain hinge region using Molecular Dynamics simulations combined with Force Distribution Analysis (FDA). While previously identified key residues involved in PGK domain closure are part of this pathway, we here fill the numerous gaps in the pathway by identifying newly uncovered residues and interesting candidates for future mutational studies. Dynamics (MD) trajectories [1922] or Elastic Network models [23,24]. However, typical timescales of allosteric transitions are in order of millisecond to seconds, while atomic simulations cover the femtosecond to microsecond range. Also, these methods focus on large-amplitude motions, while allosteric sign (...truncated)